摘要
目的 评价重组PPE65蛋白IgG抗体用于检测肺结核患者的价值.方法 将编码结核分枝杆菌PPE65蛋白的基因克隆到PET-28a载体,并在大肠埃希菌中表达,镍亲和层析和离子层析法纯化重组PPE65蛋白,透析复性和Lowry法测蛋白浓度.采用ELISA法检测144例肺结核患者、144名健康者、56例非结核肺部疾病患者血清中抗重组PPE65蛋白和重组PstS1蛋白的IgG抗体水平.用ELISA检测144例肺结核患者和97名PPD皮试阴性健康人血清IgG结果绘制ROC曲线确定临界值.分析评价重组PPE65蛋白及其与重组PstS1蛋白联合检测肺结核的敏感度、特异度、阳性预测值、阴性预测值、准确性.结果 结核分枝杆菌PPE65蛋白在大肠埃希菌中获得表达,层析纯化获得纯度为95%的重组PPE65蛋白,复性后蛋白浓度为0.5 mg/ml.重组PPE65蛋白IgG抗体的ELISA检测临界值为0.64.重组PPE65蛋白诊断肺结核的敏感度、特异度、阳性预测值、阴性预测值、准确性分别为34.7%（50/144）、93.5%（187/200）、79.4%（50/63）、66.5%（187/281）、68.9%（237/344）;重组PPE65蛋白和重组PstS1蛋白联合诊断肺结核的敏感度、特异度、阳性预测值、阴性预测值、准确性分别为59.0%（85/144）、91.0%（182/200）、82.5%（85/103）、75.5%（182/241）、77.6%（267/344）.结论 结核分枝杆菌PPE65蛋白可作为结核患者血清检测的蛋白抗原之一.PPE65蛋白和重组PstS1蛋白IgG抗体联合检测用于结核病诊断可提高敏感度.
Objective To evaluate the potential value of IgG antibodies against recombinant PPE65 protein （rPPE65） of Mycobacterium tuberculosis in serodiagnosis of tuberculosis.Methods The gene encoding PPE65 protein of M.tuberculosis was cloned into the PET-28a vector and then expressed in Escherichia coli.The rPPE65 was purified with Ni-NTA affinity and ion exchange chromatography.After dialysis renaturation, the concentration of rPPE65 was determined using Lowry assay.ELISA was used to detect the levels of specific IgG against rPPE65 and recombinant PstS1 protein （rPstS1） in sera from 144 patients with pulmonary tuberculosis （PTB patients）, 144 health controls, and 56 patients with non-tuberculosis pulmonary diseases.ROC curves were used to determine cut-off values with the results of IgG antibodies against rPPE65 and rPstS1 for 144 PTB patients and 97 controls with negative PPD skin test.The sensitivity, specificity, positive predictive value （PPV）, negative predictive value （NPV）, and accuracy of rPPE65 and the combination of rPPE65 and rPstS1 were counted.Results The PPE65 protein of M.tuberculosis was successfully expressed in E.coli. The purity and concentration of rPPE65 were 95% and 0.5 mg/ml, respectively.ROC analysis showed that the cut-off of ELISA using rPPE65 was 0.64.The sensitivity, specificity, PPV, NPV, and accuracy of rPPE65 were 34.7%（50/144）, 93.5%（187/200）, 79.4%（50/63）, 66.5%（187/287）, and 68.9%（237/344）, respectively.The sensitivity, specificity, PPV, NPV, and accuracy of the combination of rPPE65 and rPstS1 were 59.0%, 91.0%, 82.5%, 75.5%, 77.6%, respectively.Conclusions The rPPE65 of M.tuberculosis appears to be a candidate antigen for serodiagnosis of tuberculosis.Detection of IgG antibodies against the combination of rPPE65 and rPstS1 can increase the sensitivity of serological test for tuberculosis.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2011年第7期633-637,共5页
Chinese Journal of Laboratory Medicine
基金
基金项目：“十一五”国家高技术研究发展计划资助项目（2006AA022465）
全军“十一五”面上课题资助项目（06M302）
关键词
结核
肺
抗原
细菌
酶联免疫吸附测定
Tuberculosis,pulmonary
Antigens,bacterial
Enzyme-linked immunosorbent assay
